Method of nucleotide detection

ABSTRACT

The invention relates to an additive which can be added to buffers used in nucleotide detection processes and improved methods of nucleic acid sequencing using this additive. In particular the invention relates to use of the additive to improve the efficiency of fluorescence-based multiple cycle nucleic acid sequencing reactions.

FIELD OF THE INVENTION

The invention relates to an additive which can be added to buffers usedin nucleotide detection processes and improved methods of nucleic acidsequencing using this additive. In particular the invention relates touse of the additive to improve the efficiency of fluorescence-basedmultiple cycle nucleic acid sequencing reactions.

BACKGROUND TO THE INVENTION

There are known in the art methods of nucleic acid sequencing based onsuccessive cycles of incorporation of fluorescently labelled nucleicacid analogues. In such “sequencing by synthesis” or “cycle sequencing”methods the identity of the added base is determined after eachnucleotide addition by detecting the fluorescent label.

In particular, U.S. Pat. No. 5,302,509 describes a method for sequencinga polynucleotide template which involves performing multiple extensionreactions using a DNA polymerase or DNA ligase to successivelyincorporate labelled polynucleotides complementary to a template strand.In such a “sequencing by synthesis” reaction a new polynucleotide strandbased-paired to the template strand is built up in the 5′ to 3′direction by successive incorporation of individual nucleotidescomplementary to the template strand. The substrate nucleosidetriphosphates used in the sequencing reaction are labelled at the 3′position with different 3′ labels, permitting determination of theidentity of the incorporated nucleotide as successive nucleotides areadded.

The present inventors have developed methods of sequencing multiplenucleic acid molecules in parallel based on the use of arrays, whereinmultiple template molecules immobilised on the array are sequenced inparallel. Such arrays may be single molecule arrays or clustered arrays.

The inventors have observed that when performing sequencing-by-synthesisusing fluorescently labelled nucleotide analogues the brightness of theincorporated fluorophore diminishes at each cycle of nucleotideaddition. This is a particular problem when sequencing nucleic acidtemplates on arrays, and more particularly arrays comprised of clustersof surface-bound DNA. Cycles of sequencing may stop at around cycle 8-10due to loss of signal from the fluorescently labelled nucleotideanalogue incorporated into extended strand complementary to thetemplate, making it difficult to score accurately the identity of theincorporated base. Moving to a new area of the array that has not beenpreviously scanned however clearly shows that the correct base can beaccurately read. This is indicative of light-induced damage to thenucleic acid templates upon repeated exposures to the intenseillumination used to read the incorporated fluorophores.

It is known in the art to add chemical antioxidants such as ascorbicacid (vitamin C) to fluorescent imaging buffers. It has, however, neverpreviously been suggested to add such antioxidants to buffers used fordetection/imaging of fluorophores incorporated in or attached to nucleicacid. More particularly, it has never previously been suggested to useantioxidants such as ascorbate as additives in buffers used for imagingof arrays during cycles of nucleic acid sequencing.

The inventors have now observed that the presence of one or moreantioxidants in buffers used in any molecular biology techniquerequiring detection of a fluorescent moiety incorporated in or attachedto a nucleic acid, which detection includes repeated or prolongedexposure to intense illumination, is surprisingly advantageous.

DESCRIPTION OF THE INVENTION

In a first aspect the invention provides a method of detecting afluorescent moiety incorporated in or attached to a polynucleotidemolecule, wherein the method includes a detection step which requiresrepeated or prolonged exposure to intense illumination, and whereindetection of the fluorescent moiety is carried out in a buffer whichcomprises one or more antioxidants.

The invention will now be further described. In the following passagesdifferent features of the invention are defined in more detail. Eachfeature so defined may be combined with any other feature or featuresunless clearly indicated to the contrary. In particular any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

The method of the invention is useful in many techniques requiringincorporation of a fluorescently labeled nucleotide into apolynucleotide, including but not limited to sequencing reactions,polynucleotide synthesis, nucleic acid amplification, nucleic acidhybridisation assays, single nucleotide polymorphism studies, and othersuch techniques. All such uses and methods utilizing the bufferadditives of the invention in the imaging/detection steps are includedwithin the scope of the present invention.

Preferably the method is a sequencing reaction, particularly asequencing-by-synthesis reaction. In particular the method of inventionis of particular utility in a method of sequencing a template nucleicacid comprising incorporating one or more fluorescently labellednucleotides into a strand of nucleic acid complementary to said templatenucleic acid and determining the identity of the base present in one ormore of the incorporated nucleotide(s), wherein the step of determiningthe identity of the base present in the incorporated nucleotide(s) iscarried out in a buffer which comprises one or more antioxidants.

The subsequent discussion shall focus mainly on sequencing-by-synthesisreactions although it is to be understood that the invention should notbe considered as so limited.

Use of a buffer which comprises one or more antioxidants in such methodssubstantially improves performance, increasing the number of nucleotideadditions which can be accurately determined in each sequencingexperiment. The inclusion of an antioxidant as an additive in the bufferprevents the loss of signal that otherwise occurs over successive cyclesof nucleotide incorporation and allows more cycles of sequencing to beachieved using the same sequencing templates.

Preferably at least one of the one or more antioxidants is ascorbic acidor a salt, analogue, or derivative thereof.

Unless otherwise stated the terms “ascorbate” and “ascorbic acid” asused herein refer to both the L-isomer and the D-isomer, and mixtures,including racemic mixtures, thereof. Both optical isomers are capable offunctioning as antioxidants.

A preferred salt includes sodium ascorbate, e.g. sodium L-ascorbate.

There are known in the art numerous ascorbate analogues and derivativeshaving antioxidant activity which can be used in the method of theinvention. Suitable derivatives and analogues include those in which the5- and/or 6-hydroxy group is esterified or otherwise derivatised.

Alternatively, the 5- and/or 6-hydroxy group may be replaced with analternative functional group, such as halo or amino. Other alternativederivatives are those in which the 5- and/or 6-hydroxy group is absentand (i.e with a hydrogen atom in place of the hydroxyl group).Representative examples of such derivatives include, but are not limitedto, 6-O-tosyl-L-ascorbate, 5-deoxy-L-ascorbate,6-bromo-6-deoxy-L-ascorbate, 6-amino-6-deoxy-L-ascorbate, L-ascorbicacid 6-carboxylate, and 6-O-ascorbyl alkanoates such as 6-ascorbylpalmitate (palmitoyl ascorbate).

Advantageously a combination of two or more antioxidants may be presentin the buffer. Preferably, at least one of the antioxidants in suchcombinations is ascorbic acid or a salt, analogue, or derivative thereofas discussed immediately hereinbefore

Preferably the one or more antioxidants will be present in the buffer ata concentration of at least 10 mM, preferably at least 20 mM. Whilstthere is no particular upper limit, generally a concentration of 100 mM,more particularly a concentration of 50 mM, represents a preferableupper limit to the concentration of antioxidant present. For example,therefore, the one or more antioxidants may be present in the buffer ata concentration in the range of from 10 to 100 mM, preferably 20 to 50mM, more preferably about 50 mM.

The pH of the buffer is not of critical importance; the invention may beoperated at a fairly broad pH range. Generally, however, broadly neutralpH ranges are appropriate for the buffers, such as from about pH 5 toabout pH 9, preferably from pH 5.5 to 8.6, e.g. about pH 7.

The nature of the buffering agent itself is likewise not of any greatimportance: any buffering agent may be used. An example of anappropriate, indeed a preferred convenient buffering agent is tris (tris(hydroxymethyl)aminomethane). Additionally salts, e.g. sodium chlorideor any other convenient salt, for example present at a concentration inthe range of from 5-100 mM, preferably 10-70 mM, and particularly 30 mM,may be present.

An example of a particularly preferred buffer which may be used in allaspects of the invention comprises Tris (100 mM), NaCl (30 mM), sodiumascorbate (50 mM) at pH 7.

The nucleotide(s) incorporated into the strand of nucleic acidcomplementary to the template nucleic are each fluorescently labelled.The inclusion of a fluorescent label facilitatesdetection/identification of the base present in the incorporatednucleotide(s). Appropriate fluorophores are well known in the art.

The labels may be the same for each type of nucleotide, or eachnucleotide type may carry a different label. This facilitates theidentification of incorporation of a particular nucleotide. Thus, forexample modified adenine, guanine, cytosine and thymine would all haveattached a different fluorophore to allow them to be discriminated fromone another readily. When sequencing on arrays, a mixture of labelledand unlabelled nucleotides may be used.

Detectable labels such as fluorophores can be linked to nucleotides viathe base using a suitable linker. The linker may be acid labile,photolabile or contain a disulfide linkage. Preferred labels andlinkages include those disclosed in WO03/048387. Other linkages, inparticular phosphine-cleavable azide-containing linkers, may be employedin the invention as described in greater detail in WO2004/018493. Thecontents of WO 03/048387 and WO 2004/018493 are incorporated herein intheir entirety by reference.

The nucleotides described in WO2004/018493 comprise a purine orpyrimidine base and a ribose or deoxyribose sugar moiety which has aremovable blocking group covalently attached thereto, preferably at the3′O position. 3′ blocking groups are also described in WO2004/018497,the contents of which is also incorporated herein in its entirety byreference. Use of such 3′-blocked nucleotides permits controlledincorporation of nucleotides in a step-wise manner, since the presenceof a blocking group at the 3′-OH position prevents incorporation ofadditional nucleotides. The detectable label may, if desirable, beincorporated into the blocking groups as is disclosed in WO2004/018497.

Methods for detecting fluorescently labelled nucleotides generallyrequire use of incident light (e.g. laser light) of a wavelengthspecific for the fluorescent label, or the use of other suitable sourcesof illumination, to excite the fluorophore. Fluorescent light emittedfrom the fluorophore may then be detected at the appropriate wavelengthusing a suitable detection system such as for example aCharge-Coupled-Device (CCD) camera, which can optionally be coupled to amagnifying device, a fluorescent imager or a confocal microscope.

If sequencing is carried out on an array, detection of an incorporatedbase may be carried out by using a confocal scanning microscope to scanthe surface of the array with a laser, to image fluorescent labelsattached to the incorporated nucleotide(s). Alternatively, a sensitive2-D detector, such as a charge-coupled detector (CCD), can be used tovisualise the signals generated. This technique is particularly usefulwith single molecule arrays. Other techniques such as scanningnear-field optical microscopy (SNOM) are available and may be used whenimaging dense arrays. For a description of scanning near-field opticalmicroscopy, see Moyer et al., Laser Focus World 29:10, 1993. Anadditional technique that may be used is surface-specific total internalreflection fluorescence microscopy (TIRFM); see, for example, Vale etal., Nature, (1996) 380: 451-453). Suitable apparatus used for imagingpolynucleotide arrays are known in the art and the technical set-up willbe apparent to the skilled person.

Detection buffers containing antioxidants, such as sodium ascorbate,show a clear improvement (over corresponding buffers absent suchantioxidants) at preventing light-induced chemical artefacts in cyclesof sequencing-by-synthesis based on detection of fluorescently labellednucleotide analogues. The inclusion of antioxidants prevents/reduceslight-induced chemical reactions from damaging the integrity of thenucleic acid template and allows accurate determination of the identityof the incorporated base over at least 2, preferably at least 10, andmore preferably at least 16 cycles of nucleotide incorporation.Preferably from 10 to 30 and more preferably from 16 to 30 nucleotidesare successively incorporated, and identified, in the sequencingreaction.

The ability to accurately sequence 10 or more, and preferably 16 ormore, consecutive nucleotides in a sequencing reaction is a significantadvantage in applications such as genome re-alignment.

In the context of this invention the terms “sequencing reaction”,“sequencing methodology” or “method of sequencing” generally refer toany polynucleotide “sequencing-by-synthesis” reaction which involvessequential addition of one or more nucleotides to a growingpolynucleotide chain in the 5′ to 3′ direction using a polymerase inorder to form an extended polynucleotide chain complementary to thetemplate nucleic acid to be sequenced.

The identity of the base present in one or more of the addednucleotide(s) is determined in a detection or “imaging” step. Theidentity of the added base is preferably determined after eachnucleotide incorporation step. The sequence of the template may then beinferred using conventional Watson-Crick base-pairing rules. For theavoidance of doubt “sequencing” can also encompass incorporation andidentification of a single nucleotide. Determination of the identity ofa single base may be useful, for example, in the scoring of singlenucleotide polymorphisms.

The nucleic acid template to be sequenced in a sequencing reaction maybe any polynucleotide that it is desired to sequence. The nucleic acidtemplate for a sequencing reaction will typically comprise adouble-stranded region having a free 3′ hydroxyl group which serves as aprimer or initiation point for the addition of further nucleotides inthe sequencing reaction. The region of the template to be sequenced willoverhang this free 3′ hydroxyl group on the complementary strand. Theprimer bearing the free 3′ hydroxyl group may be added as a separatecomponent (e.g. a conventional oligonucleotide sequencing primer) whichhybridises to a region of the template to be sequenced. Alternatively,the primer and the template strand to be sequenced may each form part ofa partially self-complementary nucleic acid strand capable of forming anintramolecular duplex, such as for example a hairpin loop structure.Nucleotides are added successively to the free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. After each nucleotide addition the nature of the base whichhas been added may be determined, thus providing sequence informationfor the nucleic acid template.

The term “incorporation” of a nucleotide into a nucleic acid strand (orpolynucleotide) refers to joining of the nucleotide to the free 3′hydroxyl group of the nucleic acid strand via formation of aphosphodiester linkage with the 5′ phosphate group of the nucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Thenucleic acid may comprise naturally occurring and/or non-naturallyoccurring nucleotides and natural or non-natural backbone linkages.

Nucleic acid templates to be sequenced may be attached to a solidsupport via any suitable linkage method known in the art. Preferablylinkage will be via covalent attachment. If the templates are “arrayed”on a solid support then the array may take any convenient form. Thus,the method of the invention is applicable to all types of “high density”arrays, including single-molecule arrays and clustered arrays.

When referring to immobilisation of molecules (e.g. nucleic acids) to asolid support, the terms “immobilised” and “attached” are usedinterchangeably herein and both terms are intended to encompass director indirect, covalent or non-covalent attachment, unless indicatedotherwise, either explicitly or by context. In certain embodiments ofthe invention covalent attachment may be preferred, but generally allthat is required is that the molecules (e.g. nucleic acids) remainimmobilised or attached to the support under the conditions in which itis intended to use the support, for example in applications requiringnucleic acid amplification and/or sequencing.

Certain embodiments of the invention may make use of solid supportscomprised of an inert substrate or matrix (e.g. glass slides, polymerbeads etc) which is been “functionalised”, for example by application ofa layer or coating of an intermediate material comprising reactivegroups which permit covalent attachment to biomolecules, such aspolynucleotides. Examples of such supports include, but are not limitedto, polyacrylamide hydrogels supported on an inert substrate such asglass, particularly polyacrylamide hydrogels as described in WO2005/065814, the contents of which are incorporated herein in theirentirety by reference. In such embodiments, the biomolecules (e.g.polynucleotides) may be directly covalently attached to the intermediatematerial (e.g. the hydrogel) but the intermediate material may itself benon-covalently attached to the substrate or matrix (e.g. the glasssubstrate). The term “covalent attachment to a solid support” is to beinterpreted accordingly as encompassing this type of arrangement.

The method of the invention may be used for sequencing on essentiallyany type of array formed by immobilisation of nucleic acid molecules ona solid support, and more particularly any type of high-density array.However, the method of the invention is particularly advantageous in thecontext of sequencing on clustered arrays.

In multi-polynucleotide or clustered arrays distinct regions on thearray comprise multiple polynucleotide template molecules. Depending onhow the array is formed each site on the array may comprise multiplecopies of one individual polynucleotide molecule or even multiple copiesof a small number of different polynucleotide molecules (e.g. multiplecopies of two complementary nucleic acid strands).

Multi-polynucleotide or clustered arrays of nucleic acid molecules maybe produced using techniques generally known in the art. By way ofexample, WO 98/44151 and WO 00/18957 both describe methods ofsolid-phase nucleic acid amplification which allow amplificationproducts to be immobilised on a solid support in order to form arrayscomprised of clusters or “colonies” of immobilised nucleic acidmolecules. Each cluster or colony on such an array is formed from aplurality of identical immobilised polynucleotide strands and aplurality of identical immobilised complementary polynucleotide strands.The arrays so-formed are generally referred to herein as “clusteredarrays”. The nucleic acid molecules present on the clustered arraysprepared according to these methods are suitable templates forsequencing using the method of the invention. However, the invention isnot intended to use of the method in sequencing reactions carried out onclustered arrays prepared according to these specific methods.

The products of solid-phase amplification reactions such as thosedescribed in WO 98/44151 and WO 00/18957 are so-called “bridged”structures formed by annealing of pairs of immobilised polynucleotidestrands and immobilised complementary strands, both strands beingimmobilised on the solid support at the 5′ end, preferably via acovalent attachment. In order to improve the performance of sequencingon arrays comprised of such bridged structures it is preferred to removesubstantially all or at least a portion of one of the immobilisedstrands in the “bridged” structure in order to generate a template whichis at least partially single-stranded. The portion of the template whichis single-stranded will thus be available for hybridisation to asequencing primer. The process of removing all or a portion of oneimmobilised strand in a “bridged” double-stranded nucleic acid structuremay be referred to herein as “linearisation”.

It is known in the art that bridged template structures may belinearised by cleavage of one or both strands with a restrictionendonuclease and this method of linearisation may be used in the contextof the invention. A disadvantage of the use of restriction enzymes forlinearisation is that it requires the presence of a specific recognitionsequence for the enzyme at a suitable location in the bridged templatestructure. There is a risk that the same recognition sequence may appearelsewhere in the bridged structure, meaning that the enzyme may cut atone or more further sites, in addition to the intended cleavage site forlinearisation. This may be a particular problem where the bridgedstructures to be linearised are derived by solid-phase amplification oftemplates of unknown or partially unknown sequence, since it cannot bepredicted in advance whether a particular enzyme will cut within theregion of unknown sequence.

The inventors have developed a number of alternative cleavage methodsfor template linearisation which do not require the use of restrictionenzymes, as described below. Any of these methods can be used forlinearisation of “bridged” nucleic acid structures in clustered arraysformed by solid-phase amplification, and indeed for linearisation any“bridged” double-stranded nucleic acid molecule formed by annealing ofcomplementary polynucleotide strands that are attached to a solidsupport at the 5′ end, whether or not formed by solid-phaseamplification.

Therefore, another aspect the invention provides a method of generatinga template for a nucleic acid sequencing reaction comprising,

(i) providing at least one double-stranded nucleic acid molecule,wherein both strands of the double-stranded nucleic acid molecule areattached to the solid support at the 5′ end,(ii) cleaving one or both strands of the double-stranded nucleic acidmolecule, and(iii) subjecting the cleaved strand(s) to denaturing conditions toremove the portion of the cleaved strand(s) not attached to the solidsupport, thereby generating a partially or substantially single-strandedtemplate for a nucleic acid sequencing reaction,characterised in that step (ii) does not comprise cleavage with arestriction endonuclease or a nicking endonuclease.

In step (i) the double-stranded nucleic acid molecule is typically abridged nucleic acid structure produced by a solid-phase nucleic acidamplification method.

In one embodiment cleavage may occur at a cleavage site in one or bothstrands of the double-stranded nucleic acid molecule which comprises oneor more or any combination of non-natural nucleotides, ribonucleotidesor a non-nucleotide chemical modifications.

Preferred cleavage methods are as follows:

i) Chemical Cleavage

The term “chemical cleavage” encompasses any method which utilises anon-nucleic acid and non-enzymatic chemical reagent in order topromote/achieve cleavage of one or both strands of a double-strandednucleic acid molecule. If required, one or both strands of thedouble-stranded nucleic acid molecule may include one or morenon-nucleotide chemical moieties and/or non-natural nucleotides and/ornon-natural backbone linkages in order to permit chemical cleavagereaction. In a preferred embodiment the modification(s) required topermit chemical cleavage may be incorporated into an amplificationprimer used in solid-phase nucleic acid amplification.

In a preferred but non-limiting embodiment one strand of thedouble-stranded nucleic acid molecule (or the amplification primer fromwhich this strand is derived if formed by solid-phase amplification) mayinclude a diol linkage which permits cleavage by treatment withperiodate (e.g. sodium periodate). It will be appreciate that more thanone diol can be included at the cleavage site.

Diol linker units based on phosphoamidite chemistry suitable forincorporation into polynucleotide chains are commercially available fromFidelity systems Inc. (Gaithersburg, Md., USA). One or more diol unitsmay be incorporated into a polynucleotide using standard methods forautomated chemical DNA synthesis. Hence, oligonucleotide primersincluding one or more diol linkers can be conveniently prepared bychemical synthesis.

In order to position the diol linker at an optimum distance from thesolid support one or more spacer molecules may be included between thediol linker and the site of attachment to the solid support. The spacermolecule may be a non-nucleotide chemical moiety. Suitable spacer unitsbased on phosphoamidite chemistry for use in conjunction with diollinkers are also supplied by Fidelity Systems Inc. One suitable spacerfor use with diol linkers is the spacer denoted arm 26, identified inthe accompanying examples. To enable attachment to a solid support atthe 5′ end of the polynucleotide strand arm 26 may be modified toinclude a phosphorothioate group. The phosphorothioate group can easilybe attached during chemical synthesis of a “polynucleotide” chainincluding the spacer and diol units.

Other spacer molecules could be used as an alternative to arm 26. Forexample, a stretch of non-target “spacer” nucleotides may be included.Typically from 1 to 20, more preferably from 1 to 15 or from 1 to 10,and more particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10 spacer nucleotidesmay be included. Most preferably 10 spacer nucleotides will bepositioned between the point of attachment to the solid support and thediol linker. It is preferred to use polyT spacers, although othernucleotides and combinations thereof can be used. In one preferredembodiment the primer may include 10T spacer nucleotides.

The diol linker is cleaved by treatment with a “cleaving agent”, whichcan be any substance which promotes cleavage of the diol. The preferredcleaving agent is periodate, preferably aqueous sodium periodate(NaIO₄). Following treatment with the cleaving agent (e.g. periodate) tocleave the dial, the cleaved product may be treated with a “cappingagent” in order to neutralise reactive species generated in the cleavagereaction. Suitable capping agents for this purpose include amines, suchas ethanolamine. Advantageously, the capping agent (e.g. ethanolamine)may be included in a mixture with the cleaving agent (e.g. periodate) sothat reactive species are capped as soon as they are formed.

The combination of a diol linkage and cleaving agent (e.g. periodate) toachieve cleavage of one strand of a double-stranded nucleic acidmolecule is preferred for linearisation of nucleic acid molecules onsolid supported polyacrylamide hydrogels because treatment withperiodate is compatible with nucleic acid integrity and with thechemistry of the hydrogel surface. However, utility of diollinkages/periodate as a method of linearisation is not limited topolyacrylamide hydrogel surfaces but also extends to linearisation ofnucleic acids immobilised on other solid supports and surfaces,including supports coated with functionalised silanes (etc).

In a further embodiment, the strand to be cleaved (or the amplificationprimer from which this strand is derived if prepared by solid-phaseamplification) may include a disulphide group which permits cleavagewith a chemical reducing agent, e.g. Tris (2-carboxyethyl)-phosphatehydrochloride (TCEP).

ii) Cleavage of Abasic Sites in a Double-Stranded Molecule

An “abasic site” is defined as a nucleoside position in a polynucleotidechain from which the base component has been removed. Abasic sites canoccur naturally in DNA under physiological conditions by hydrolysis ofnucleoside residues, but may also be formed chemically under artificialconditions or by the action of enzymes. Once formed, abasic sites may becleaved (e.g. by treatment with an endonuclease or other single-strandedcleaving enzyme, exposure to heat or alkali), providing a means forsite-specific cleavage of a polynucleotide strand.

In a preferred but non-limiting embodiment an abasic site may be createdat a pre-determined position on one strand of a double-strandedpolynucleotide and then cleaved by first incorporating deoxyuridine (U)at a pre-determined cleavage site in one strand of the double-strandednucleic acid molecule. This can be achieved, for example, by including Uin one of the primers used for preparation of the double-strandednucleic acid molecule by solid-phase PCR amplification. The enzymeuracil DNA glycosylase (UDG) may then be used to remove the uracil base,generating an abasic site on one strand. The polynucleotide strandincluding the abasic site may then be cleaved at the abasic site bytreatment with endonuclease (e.g EndoIV endonuclease, AP lyase, FPGglycosylase/AP lyase, EndoVIII glycosylase/AP lyase), heat or alkali.

Abasic sites may also be generated at non-natural/modifieddeoxyribonucleotides other than deoxyuridine and cleaved in an analogousmanner by treatment with endonuclease, heat or alkali. For example,8-oxo-guanine can be converted to an abasic site by exposure to FPGglycosylase. Deoxyinosine can be converted to an abasic site by exposureto AlkA glycosylase. The abasic sites thus generated may then becleaved, typically by treatment with a suitable endonuclease (e.g.EndoIV, AP lyase). If the non-natural/modified nucleotide is to beincorporated into an amplification primer for use in solid-phaseamplification, then the non-natural/modified nucleotide should becapable of being copied by the polymerase used for the amplificationreaction.

In one embodiment, the molecules to be cleaved may be exposed to amixture containing the appropriate glycosylase and one or more suitableendonucleases. In such mixtures the glycosylase and the endonucleasewill typically be present in an activity ratio of at least about 2:1.

This method of cleavage has particular advantages in relation to thecreation of templates for nucleic acid sequencing. In particular,cleavage at an abasic site generated by treatment with a glycosylasesuch as UDG generates a free 3′ hydroxyl group on the cleaved strandwhich can provide an initiation point for sequencing a region of thecomplementary strand. Moreover, if the starting double-stranded nucleicacid contains only one cleavable (e.g. uracil) base on one strand then asingle “nick” can be generated at a unique position in this strand ofthe duplex. Since the cleavage reaction requires a residue, e.g.deoxyuridine, which does not occur naturally in DNA, but is otherwiseindependent of sequence context, if only one non-natural base isincluded there is no possibility of glycosylase-mediated cleavageoccurring elsewhere at unwanted positions in the duplex. In contrast,were the double-stranded nucleic acid to be cleaved with a “nicking”endonuclease that recognises a specific sequence, there is a possibilitythat the enzyme may create nicks at “other” sites in the duplex (inaddition to the desired cleavage site) if these possess the correctrecognition sequence. This could present a problem if nicks are createdin the strand it is intended to sequence rather than the strand thatwill be fully or partially removed to create the sequencing template andis a particular risk if the target portion of the double-strandednucleic acid molecule is of unknown sequence.

The fact that there is no requirement for the non-natural (e.g. uracil)residue to be located in a detailed sequence context in order to providea site for cleavage using this approach is itself advantageous. Inparticular, if the cleavage site is to be incorporated into anamplification primer to be used in the production of a clustered arrayby solid-phase amplification, it is necessarily only to replace onenatural nucleotide (e.g. T) in the primer with a non-natural nucleotide(e.g. U) in order to enable cleavage. There is no need to engineer theprimer to include a restriction enzyme recognition sequence of severalnucleotides in length. Oligonucleotide primers including U nucleotides,and the other non-natural nucleotides listed above, can easily beprepared using conventional techniques and apparatus for chemicalsynthesis of oligonucleotides.

Another advantage gained by cleavage of abasic sites in adouble-stranded molecule generated by action of UDG on uracil is thatthe first base incorporated in a “sequencing-by-synthesis” reactioninitiating at the free 3′ hydroxyl group formed by cleavage at such asite will always be T. Hence, if the double-stranded nucleic acidmolecule forms part of a clustered array comprised of many suchmolecules, all of which are cleaved in this manner to produce sequencingtemplates, then the first base universally incorporated across the wholearray will be T. This can provide a sequence-independent assay forcluster intensity at the start of a sequencing “run”.

iii) Cleavage of Ribonucleotides

Incorporation of one or more ribonucleotides into a polynucleotidestrand which is otherwise comprised of deoxyribonucleotides (with orwithout additional non-nucleotide chemical moieties, non-natural basesor non-natural backbone linkages) can provide a site for cleavage usinga chemical agent capable of selectively cleaving the phosphodiester bondbetween a deoxyribonucleotide and a ribonucleotide or using aribonuclease (RNAse). Therefore, sequencing templates can be produced bycleavage of one strand of a “bridged” structure at a site containing oneor more consecutive ribonucleotides using such a chemical cleavage agentor an RNase. Preferably the strand to be cleaved contains a singleribonucleotide to provide a site for chemical cleavage.

Suitable chemical cleavage agents capable of selectively cleaving thephosphodiester bond between a deoxyribonucleotide and a ribonucleotideinclude metal ions, for example rare-earth metal ions (especially La³⁺,particularly Tm³⁺, Yb³⁺ or Lu³⁺ (Chen et al. Biotechniques. 2002, 32:518-520; Komiyama et al. Chem. Commun. 1999, 1443-1451)), Fe(3) orCu(3), or exposure to elevated pH, e.g. treatment with a base such assodium hydroxide. By “selective cleavage of the phosphodiester bondbetween a deoxyribonucleotide and a ribonucleotide” is meant that thechemical cleavage agent is not capable of cleaving the phosphodiesterbond between two deoxyribonucleotides under the same conditions.

The base composition of the ribonucleotide(s) is generally not material,but can be selected in order to optimise chemical (or enzymatic)cleavage. By way of example, rUMP or rCMP are generally preferred ifcleavage is to be carried out by exposure to metal ions, especially rareearth metal ions.

The ribonucleotide(s) will typically be incorporated into one strand ofa “bridged” double-stranded nucleic acid molecule (or the amplificationprimer from which this strand is derived if prepared by solid-phaseamplification), and may be situated in a region of the bridged structurewhich is single-stranded when the two complementary strands of thedouble-stranded molecule are annealed (i.e. in a 5′ overhangingportion). If the double-stranded nucleic acid molecule is prepared bysolid-phase PCR amplification using forward and reverse amplificationprimers, one of which contains at least one ribonucleotide, the standardDNA polymerase enzymes used for PCR amplification are not capable ofcopying ribonucleotide templates. Hence, the PCR products will containan overhanging 5′ region comprising the ribonucleotide(s) and anyremainder of the amplification primer upstream of the ribonucleotide(s).

The phosphodiester bond between a ribonucleotide and adeoxyribonucleotide or between two ribonucleotides may also be cleavedby an RNase. Any endocytic ribonuclease of appropriate substratespecificity can be used for this purpose. If the ribonucleotide(s) arepresent in a region which is single-stranded when the two complementarystrands of the double-stranded molecule are annealed (i.e. in a 5′overhanging portion), then the RNase will be an endonuclease which hasspecificity for single strands containing ribonucleotides. For cleavagewith ribonuclease it is preferred to include two or more consecutiveribonucleotides, and preferably from 2 to 10 or from 5 to 10 consecutiveribonucleotides. The precise sequence of the ribonucleotides isgenerally not material, except that certain RNases have specificity forcleavage after certain residues. Suitable RNases include, for example,RNaseA, which cleaves after C and U residues. Hence, when cleaving withRNaseA the cleavage site must include at least one ribonucleotide whichis C or U.

Polynucleotides incorporating one or more ribonucleotides can be readilysynthesised using standard techniques for oligonucleotide chemicalsynthesis with appropriate ribonucleotide precursors. If thedouble-stranded nucleic acid molecule is prepared by solid-phase nucleicacid amplification, then it is convenient to incorporate one or moreribonucleotides into one of the primers to be used for the amplificationreaction.

iv) Photochemical Cleavage

The term “photochemical cleavage” encompasses any method which utiliseslight energy in order to achieve cleavage of one or both strands of thedouble-stranded nucleic acid molecule.

A site for photochemical cleavage can be provided by a non-nucleotidechemical spacer unit in one of the strands of the double-strandedmolecule (or the amplification primer from which this strand is derivedif prepared by solid-phase amplification). Suitable photochemicalcleavable spacers include the PC spacer phosphoamidite(4-(4,4′-Dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite)supplied by Glen Research, Sterling, Va., USA (cat number 10-4913-XX)which has the structure:

The spacer unit can be cleaved by exposure to a UV light source.

This spacer unit can be attached to the 5′ end of a polynucleotide,together with a thiophosphate group which permits attachment to a solidsurface, using standard techniques for chemical synthesis ofoligonucleotides.

Conveniently, this spacer unit can be incorporated into a forward orreverse amplification primer to be used for synthesis of aphotocleavable double-stranded nucleic acid molecule by solid-phaseamplification.

v) Cleavage of Hemimethylated DNA

Site-specific cleavage of one strand of a double-stranded nucleic acidmolecule may also be achieved by incorporating one or more methylatednucleotides into this strand and then cleaving with an endonucleaseenzyme specific for a recognition sequence including the methylatednucleotide(s).

The methylated nucleotide(s) will typically be incorporated in a regionof one strand of the double-stranded nucleic acid molecule having acomplementary stretch of non-methylated deoxyribonucleotides on thecomplementary strand, such that annealing of the two strands produces ahemimethylated duplex structure. The hemimethylated duplex may then becleaved by the action of a suitable endonuclease. For the avoidance ofdoubt, enzymes which cleave such hemimethylated target sequences are notto be considered as “restriction endonucleases” excluded from the scopeof the second aspect of the invention, but rather are intended to formpart of the subject-matter of the invention.

Polynucleotides incorporating one or methylated nucleotides may beprepared using standard techniques for automated DNA synthesis, usingappropriately methylated nucleotide precursors. If the double-strandednucleic acid molecule is prepared by solid-phase nucleic acidamplification, then it is convenient to incorporate one or moremethylated nucleotides into one of the primers to be used for theamplification reaction.

vi) PCR Stoppers

In another embodiment of the invention the double-stranded nucleic acidmay be prepared by solid-phase amplification using forward and reverseprimers, one of which contains a “PCR stopper”. A “PCR stopper” is anymoiety (nucleotide or non-nucleotide) which prevents read-through of thepolymerase used for amplification, such that it cannot copy beyond thatpoint. The result is that amplified strands derived by extension of theprimer containing the PCR stopper will contain a 5′ overhanging portion.This 5′ overhang (other than the PCR stopper itself) may be comprised ofnaturally occurring deoxyribonucleotides, with predominantly naturalbackbone linkages, i.e. it may simply be a stretch of single-strandedDNA. The molecule may then be cleaved in the 5′ overhanging region withthe use of a cleavage reagent (e.g. an enzyme) which is selective forcleavage of single-stranded DNA but not double stranded DNA, for examplemung bean nuclease.

The PCR stopper may be essentially any moiety which preventsread-through of the polymerase to be used for the amplificationreaction. Suitable PCR stoppers include, but are not limited to,hexaethylene glycol (HEG), abasic sites, and any non-natural or modifiednucleotide which prevents read-through of the polymerase, including DNAanalogues such as peptide nucleic acid (PNA).

Stable abasic sites can be introduced during chemical oligonucleotidesynthesis using appropriate spacer units containing the stable abasicsite. By way of example, abasic furan(5′-O-Dimethoxytrityl-1′,2′-Dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)spacers commercially available from Glen Research, Sterling, Va., USA,can be incorporated during chemical oligonucleotide synthesis in orderto introduce an abasic site. Such a site can thus readily be introducedinto an oligonucleotide primer to be used in solid-phase amplification.If an abasic site is incorporated into either forward or reverseamplification primer the resulting amplification product will have a 5′overhang on one strand which will include the abasic site (insingle-stranded form). The single-stranded abasic site may then becleaved by the action of a suitable chemical agent (e.g. exposure toalkali) or an enzyme (e.g. AP-endonuclease VI, Shida el al. NucleicAcids Research, 1996, Vol. 24, 4572-4576).

vii) Cleavage of Peptide Linker

A cleavage site can also be introduced into one strand of thedouble-stranded nucleic molecule by preparing a conjugate structure inwhich a peptide molecule is linked to one strand of the nucleic acidmolecule (or the amplification primer from which this strand is derivedif prepared by solid-phase amplification). The peptide molecule cansubsequently be cleaved by a peptidase enzyme of the appropriatespecificity, or any other suitable means of non-enzymatic chemical orphotochemical cleavage. Typically, the conjugate between peptide andnucleic acid will be formed by covalently linking a peptide to onestrand only of the double-stranded nucleic acid molecule, with thepeptide portion being conjugated to the 5′ end of this strand, adjacentto the point of attachment to the solid surface. If the double-strandednucleic acid is prepared by solid-phase amplification, the peptideconjugate may be incorporated at the 5′ end of one of the amplificationprimers. Obviously the peptide component of this primer will not becopied during PCR amplification, hence the “bridged” amplificationproduct will include a cleavable 5′ peptide “overhang” on one strand.

Conjugates between peptides and nucleic acids wherein the peptide isconjugated to the 5′ end of the nucleic acid can be prepared usingtechniques generally known in the art. In one such technique the peptideand nucleic acid components of the desired amino acid and nucleotidesequence can be synthesised separately, e.g. by standard automatedchemical synthesis techniques, and then conjugated in aqueous/organicsolution. By way of example, the OPeC™ system commercially availablefrom Glen Research is based on the “native ligation” of an N-terminalthioester-functionalized peptide to a 5′-cysteinyl oligonucleotide.Pentafluorophenyl S-benzylthiosuccinate is used in the final couplingstep in standard Fmoc-based solid-phase peptide assembly. Deprotectionwith trifluoroacetic acid generates, in solution, peptides substitutedwith an N-terminal S-benzylthiosuccinyl group.O-trans-4-(N-a-Fmoc-S-tert-butylsulfenyl-1-cysteinyl)aminocyclohexylO-2-cyanoethyl-N,N-diisopropylphosphoramidite is used in the finalcoupling step in standard phosphoramidite solid-phase oligonucleotideassembly. Deprotection with aqueous ammonia solution generates insolution 5′-S-tert-butylsulfenyl-L-cysteinyl functionalizedoligonucleotides. The thiobenzyl terminus of the Modified Peptide isconverted to the thiophenyl analogue by the use of thiophenol, whilstthe Modified Oligonucleotide is reduced using thetris(carboxyethyl)phosphine. Coupling of these two intermediates,followed by the “native ligation” step, leads to formation of theOligonucleotide-Peptide Conjugate.

The conjugate strand containing peptide and nucleic acid can becovalently attached to a solid support using any suitable covalentlinkage technique known in the art which is compatible with the chosensurface. If the peptide/nucleic acid conjugate structure is anamplification primer to be used for solid-phase PCR amplification,attachment to the solid support must leave the 3′ end of the nucleicacid component free.

The peptide component can be designed to be cleavable by any chosenpeptidase enzyme, of which many are known in the art. The nature of thepeptidase is not particularly limited, it is necessary only for thepeptidase to cleave somewhere in the peptide component. Similarly, thelength and amino acid sequence of the peptide component is notparticularly limited except by the need to be “cleavable” by the chosenpeptidase.

The length and precise sequence of the nucleic acid component is alsonot particularly limited, it may be of any desired sequence. If thenucleic acid component is to function as a primer in solid-phase PCR,then its length and nucleotide sequence will be selected to enableannealing to the template to be amplified.

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove the portion(s) of the cleaved strand(s)that are not attached to the solid support. Suitable denaturingconditions will be apparent to the skilled reader with reference tostandard molecular biology protocols (Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor Laboratory Press, NY; Current Protocols, edsAusubel et al.).

Denaturation (and subsequent re-annealing of the cleaved strands)results in the production of a sequencing template which is partially orsubstantially single-stranded. A sequencing reaction may then beinitiated by hybridisation of a sequencing primer to the single-strandedportion of the template.

The buffer additives and methods of the invention may be utilised in thenucleic acid sequencing methods described in WO 98/44152 and WO00/18957, the contents of which are incorporated herein by reference.Both of these published International applications describe methods ofparallel sequencing of multiple templates located at distinct locationson a solid support, and in particular sequencing of “clustered” arrays.The methods described therein, and indeed any other known method ofsequencing nucleic acid clusters may be adapted according to theinvention simply by including one or more antioxidants as additives inthe buffer used for the detection/imaging steps.

As aforesaid, the use of antioxidant buffer additives in the detectionsteps has particular advantages in the context of sequencing onclustered arrays using fluorescently labelled nucleotide analogues.Nevertheless, the method of the invention may also be used in thecontext of sequencing templates on single molecule arrays of nucleicacid templates.

Single molecule arrays are generally formed by immobilisation of asingle polynucleotide molecule at each discrete site that is detectableon the array. Single-molecule arrays comprised of nucleic acid moleculesthat are individually resolvable by optical means and the use of sucharrays in sequencing are described, for example, in WO 00/06770. Singlemolecule arrays comprised of individually resolvable nucleic acidmolecules including a hairpin loop structure are described in WO01/57248. The method of the invention is suitable for sequencingtemplate molecules on single molecule arrays prepared according to thedisclosures of WO 00/06770 or WO 01/57248.

The fluorescent moiety may be attached to a nucleic acid via anysuitable covalent or non-covalent linkage. For example, the fluorescentmoiety may be attached to an oligonucleotide primer or probe which ishybridised to a target nucleic acid molecule.

Practical application of buffer additives of the invention may thereforeextend to any nucleic acid detection technique which requires the use offluorescent labels.

In a second aspect the invention provides a buffer for use indetermining the identity of the base present in nucleotide(s)incorporated in a method of sequencing according to the first aspect ofthe invention or for use in detection of a fluorescent moietyincorporated in or attached to a polynucleotide molecule according tothe second aspect of the invention, the buffer comprising one or moreantioxidants in a concentration of at least 20 mM.

Again, the preferred antioxidants are as described in connection withthe first aspect of the invention.

The buffers according to the invention will generally be ofsubstantially similar composition to the detection/imaging buffer(s)typically used in the chosen detection/imaging technique, except for theaddition of the antioxidant component(s).

The buffer may contain other reaction components such as enzymes, enzymecofactors, dNTPS etc if the presence of these components is compatiblewith the particular detection/imaging technique for which the buffer isintended to be used. For methods involving nucleic acid synthesis, suchas sequencing-by-synthesis, the same reaction buffer may be used for thenucleotide incorporation steps and for the detection steps, with nointermediate washing step. In this case the buffer may also comprise oneor more nucleotides required for the nucleic synthesis reaction and alsoa suitable polymerase enzyme.

Buffers according to the invention can be supplied as liquidconcentrates requiring dilution prior to use. Buffers may also besupplied in the form of buffer tablets or solid “concentrates” to bedissolved in a suitable solvent prior to use in order to form the finalbuffer according to the invention. Such formats may be more convenientfor supply. Buffer concentrates or tablets may be supplied together withinstructions setting out how the buffer is to be diluted prior to use.In the case of buffer concentrates and buffer tablets the amount ofantioxidant present in the buffer refers to the amount present in thefinal buffer as it is correctly diluted or made up prior to use.

In a third aspect the invention provides a kit for use in a method ofsequencing according to the first aspect of the invention comprising:

one or more nucleotides;

an enzyme capable of catalysing incorporation of said nucleotides into anucleic acid strand complementary to a nucleic acid template to besequenced; and

a buffer according to the present invention or a supply ofantioxidant(s) suitable for preparing a buffer according to the presentinvention.

The nucleotide(s) will preferably be fluorescent labelled nucleotides,as described in connection with the method of the invention. Thenucleotides may be supplied in concentrated form for dilution prior touse and may be supplied in “reagent mixes” with other reactioncomponents required for the sequencing reaction.

The enzyme may be any suitable enzyme capable of incorporating thechosen nucleotides into a nucleic acid strand. Many suitable polymeraseenzymes are known in the art and are available commercially. Inparticular, the kit may include a modified polymerase as described inthe applicant's co-pending International Patent ApplicationPCT/GB04/003891.

The buffer according to the invention may be conveniently supplied inthe kit concentrated or solid form as described above. Alternatively,the kit may simply contain a concentrated supply of antioxidant(s) to beadded into a suitable detection/imaging buffer supplied by the end userprior to use.

The invention will be further understood with reference to the followingnon-limiting experimental examples, together with the accompanyingdrawings in which:

FIG. 1 illustrates the sequence of a template DNA molecule with lambdaFfragment. The sequences and primer binding sites for the primers P7, P5and #562 are also shown.

FIG. 2 shows representative fluorescent images of clustered arrays onthe 7th cycle of nucleotide incorporation using imaging buffer withoutascorbate (3a) or with 50 mM ascorbate (3b).

EXAMPLE 1 Sequencing on Arrays Prepared on Silane Coated

Glass Chips

1) Formation of Nucleic Acid Clusters

Solid-phase amplification was carried out in 8 channel glass chips suchas those provided by Micronit (Twente, Nederland) or IMT (Neuchatel,Switzerland) coated with aminopropyltriethoxysilane derivatised with1,3,5-benzenetriacetic acid (BTA). The experimental conditions andprocedures are readily applicable to other solid supports.

The reaction steps of the coating procedure are summarised as follows:

Step 1: Conversion of Glass to Amine-Terminated Glass

Step 2: Preparation of Active Ester

Step 3: Conversion of Amine-Terminated Glass to Carboxylated Glass

The 8 channel glass chips were pre-treated and silanized with 5%aminopropyltriethoxysilane, as described in the materials and methods ofWO 00/18957 (incorporated herein by reference).

Ester activation of BTA was carried out by reacting the followingcomponents:

1,3,5-benzenetriacetic acid (BTA) 60.5 mg Fluka 17383

N-Hydroxysuccinimide (NHS) 99.5 mg Fluka: 56480N,N″-Dicyclohexylcarbodiimide (DCC) 149.6 mg Fluka: 36650

N-ethyldiisopropylamine (DIEA) 41.2 ill Perkin Elmer DMF, anhydrous onmolecular sieves SDS 0341021

The silanized glass channels were carboxylated by treatment with theactivated BTA ester then washed with DMF, ethanol, water, 5% NaHCO₃pH8.8 and water. The chips were dried under pure nitrogen and storedprior to use.

The BTA coated chips were grafted with P5 and P7 oligonucleotide primersin a 1:1 ratio. The sequences of the P5 and P7 primers are as shown inFIG. 1.

Grafting was carried out for 30 minutes at 50° C. in a grafting solution(70 μl per channel) containing 0.5 μM of each primer, 10 mM carbodiimideand 10 mM 1-methyl-imadazol.

Clusters of immobilized DNA were formed by amplification on a solidsupport substantially as described in International patent applicationWO 00/18957. Amplification was carried out using a lambdaF template andprimers P7 and P5 as shown in FIG. 1.

2) Linearisation

This procedure implies that one of the two primers used for thegeneration of thermocycled DNA colonies contains a disulfide groupbetween the amino group necessary for the binding of the primer to thesurface and its first nucleotide. This disulfide bond in the P5 primermay be cleaved using Tris (2-carboxyethyl)-phosphate hydrochloride(TCEP).

Linearization was carried out at room temperature in a TCEP/Trissolution.

Aliquots of pre-weighted TCEP (about 10 mg per aliquot) are stored at 4°C. Powder must be dissolved in Tris-HCl 100 mM pH 7.5 to get a finalTCEP concentration of 14.3 mg/ml (corresponding to 50 mM).

TCEP is sensitive to oxidation.

Templates to be linearised are typically exposed to TCEP/Tris for 30minutes then washed with 0.1×SSC-0.1% Tween, then with 5×SSC.

3) Thermal Dehybridisation

Thermal denaturation or de-hybridization of the colonies was carried outin stringent buffer (TE). Temperature is ramped 0.5° C./sec to 97.5° C.and held at 97.5° C. for 2 minutes 30 seconds.

4) Hybridisation of Sequencing Primer

The procedure begins by a heating step in a stringent buffer (TE) toensure complete denaturation of the colonies prior to hybridisation ofthe primer.

Hybridization was carried out in 5×SSC, using an oligonucleotide dilutedto a final concentration of 500 nM. This solution should be preparedjust before use, especially when fluorophore-labelled oligonucleotidesare used.

Typical temperature cycling profile is as follows:

MJ-Research Thermocycler program set:

-   -   (Control method: block)    -   1—0.5° C./sec to 97.5° C.    -   2—97.5° C. for 2:30    -   3—97.5° C. for 0:02        -   —0.1° C. per cycle    -   4—Go to 3 for 574 times    -   5—40° C. for 15:00    -   6—End        5) Long Read Sequencing Protocol on lamdaF Template

Oligo number 562 was hybridised to the linearised clusters prepared asdescribed above at 500 nM. Nucleotide sequences of the template andprimers are shown in FIG. 1.

Sequencing was carried out using modified nucleotides prepared asdescribed in International patent application WO 2004/018493, andlabelled with four different commercially available fluorophores(Molecular Probes Inc.).

A mutant 9° N polymerase enzyme (an exo-variant including the triplemutation L408Y/Y409A/P410V and C223S) was used for the nucleotideincorporation steps.

Enzyme mix (enzymology buffer above plus 50 μg/ml YAV exo-C223S, and 1μM each of the four labelled modified nucleotides) was applied to theclustered templates, typically for 2 min 30 s, and heated to 45° C.

Templates were maintained at 45° C. for 30 min, cooled to 20° C. andwashed for 5 min with enzymology buffer, then 5 min with 5×SSC.Templates were then exposed to one of two imaging buffers:“non-ascorbate buffer” (100 mM Tris pH7.0, 30 mM NaCl), or ascorbatescan buffer (100 mM Tris pH7.0, 30 mM NaCl, 50 mM sodium ascorbate(freshly dissolved, filtered).

Templates were scanned in 4 colours.

Templates were then exposed to cycles of TCEP treatment and enzymologyas follows:

TCEP Treatment

-   -   0.1M Tris pH 7.4 for 390 s    -   Heat to 45° C.    -   TCEP (100 mM in 0.1 M Tris pH 7.4) for 390 s    -   Wait for 30 min, flushing for 20 s every 10 min    -   Cool to 20° C.    -   Enzymology buffer for 390 s    -   5×SSC buffer for 390 s    -   “non ascorbate buffer” (100 mM Tris pH 7.0, 30 mM NaCl) for 330        s OR “ascorbate buffer” (100 mM Tris pH 7.0, 30 mM NaCl, 50 mM        sodium ascorbate) for 330 s    -   9. Scan in 4 colours

Enzymology Cycle

-   -   Enzymology buffer for 390 s    -   Heat to 45° C.    -   Enzyme mix for 390 s    -   Wait for 30 min, flushing for 20 s every 10 min    -   Cool to 20° C.    -   Enzymology buffer for 390 s    -   5×SSC buffer for 390 s    -   “non ascorbate buffer” (100 mM Tris pH 7.0, 30 mM NaCl) for 330        s, scan in 4 colours    -   “DMSO buffer” (100 mM Tris pH 7.0, 30 mM NaCl, 50 mM sodium        ascorbate) for 330 s, scan 4 colours

In total, 25 cycles of data were collected.

A clear difference between the number of clusters detected at each cyclecould be seen. The non ascorbate buffer resulted in a loss of signal foreach cluster, and a loss of the number of detectable clusters, such thatafter seven cycles the sequencing process was no longer working. Usingthe ascorbate buffer, at least 18 cycles of sequencing could beachieved.

The slides were scanned on a white light epifluorescence microscope setup with four separate excitation and emission channels appropriate tothe fluorophores.

Representative results are illustrated in FIG. 2.

Example 2 Sequencing on Arrays Prepared on a Hydrogel Coated GlassSurface

The solid supports used in this experiment were 8-channel glass chipssuch as those provided by Micronit (Twente, Nederland) or IMT(Neuchatel, Switzerland). However, the experimental conditions andprocedures are readily applicable to other solid supports.

Acrylamide Coating of Glass Chips

Chips were washed as follows: neat Decon for 30 min, milliQ H₂O for 30min, NaOH 1N for 15 min, milliQ H₂O for 30 min, HCl 0.1N for 15 min,milliQ H₂O for 30 min.

Polymer Solution Preparation

For 10 ml of 2% polymerisation mix.

-   -   10 ml of 2% solution of acrylamide in milliQ H₂O    -   165 μl of a 100 mg/ml N-(5-bromoacetamidylpentyl) acrylamide        (BRAPA) solution in DMF (23.5 mg in 235 μl DMF)    -   11.5 μl of TEMED    -   100 μl of a 50 mg/ml solution of potassium persulfate in milliQ        H₂O (20 mg in 400 μl H₂O)

The 10 ml solution of acrylamide was first degassed with argon for 15min. The solutions of BRAPA, TEMED and potassium persulfate weresuccessively added to the acrylamide solution. The mixture was thenquickly vortexed and immediately used. Polymerization was then carriedout for 1 h 30 at RT. Afterwards the channels were washed with milliQH₂O for 30 min. The slide are then dried by flushing argon through theinlets and stored under low pressure in a dessicator.

Synthesis of N-(5-bromoacetamidylpentyl) acrylamide (BRAPA)

N-Boc-1,5-diaminopentane toluene sulfonic acid was obtained fromNovabiochem. The bromoacetyl chloride and acryloyl chloride wereobtained from Fluka. All other reagents were Aldrich products.

To a stirred suspension of N-Boc-1,5-diaminopentane toluene sulfonicacid (5.2 g, 13.88 mmol) and triethylamine (4.83 ml, 2.5 eq) in THF (120ml) at 0° C. was added acryloyl chloride (1.13 ml, 1 eq) through apressure equalized dropping funnel over a one hour period. The reactionmixture was then stirred at room temperature and the progress of thereaction checked by TLC (petroleum ether: ethyl acetate 1:1). After twohours, the salts formed during the reaction were filtered off and thefiltrate evaporated to dryness. The residue was purified by flashchromatography (neat petroleum ether followed by a gradient of ethylacetate up to 60%) to yield 2.56 g (9.98 mmol, 71%) of product 2 as abeige solid. ¹H NMR (400 MHz, d₆-DMSO): 1.20-1.22 (m, 2H, CH₂),1.29-1.43 (m, 13H, tBu, 2×CH₂), 2.86 (q, 2H, J=6.8 Hz and 12.9 Hz, CH₂),3.07 (q, 2H, J=6.8 Hz and 12.9 Hz, CH₂), 5.53 (dd, 1H, J=2.3 Hz and 10.1Hz, CH), 6.05 (dd, 1H, J=2.3 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH), 6.77 (t, 1H, J=5.3 Hz, NH), 8.04 (bs, 1H, NH). Mass(electrospray+) calculated for C₁₃H₂₄N₂O₃ 256. found 279 (256+Na⁺).

Product 2 (2.56 g, 10 mmol) was dissolved in trifluoroaceticacid:dichloromethane (1:9, 100 ml) and stirred at room temperature. Theprogress of the reaction was monitored by TLC (dichloromethane:methanol9:1). On completion, the reaction mixture was evaporated to dryness, theresidue co-evaporated three times with toluene and then purified byflash chromatography (neat dichloromethane followed by a gradient ofmethanol up to 20%). Product 3 was obtained as a white powder (2.43 g, 9mmol, 90%). ¹H NMR (400 MHz, D₂O): 1.29-1.40 (m, 2H, CH₂), 1.52 (quint.,2H, J=7.1 Hz, CH₂), 1.61 (quint., 2H, J=7.7 Hz, CH₂), 2.92 (t, 2H, J=7.6Hz, CH₂), 3.21 (t, 2H, J=6.8 Hz, CH₂), 5.68 (dd, 1H, J=1.5 Hz and 10.1Hz, CH), 6.10 (dd, 1H, J=1.5 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH). Mass (electrospray+) calculated for C₈H₁₆N₂O 156.found 179 (156+Na⁺).

To a suspension of product 3 (6.12 g, 22.64 mmol) and triethylamine(6.94 ml, 2.2 eq) in THF (120 ml) was added bromoacetyl chloride (2.07ml, 1.1 eq), through a pressure equalized dropping funnel, over a onehour period and at −60° C. (cardice and isopropanol bath in a dewar).The reaction mixture was then stirred at room temperature overnight andthe completion of the reaction was checked by TLC(dichloromethane:methanol 9:1) the following day. The salts formedduring the reaction were filtered off and the reaction mixtureevaporated to dryness. The residue was purified by chromatography (neatdichloromethane followed by a gradient of methanol up to 5%). 3.2 g(11.55 mmol, 51%) of the product 1 (BRAPA) were obtained as a whitepowder. A further recrystallization performed in petroleum ether:ethylacetate gave 3 g of the product 1. ¹H NMR (400 MHz, d₆-DMSO): 1.21-1.30(m, 2H, CH₂), 1.34-1.48 (m, 4H, 2×CH₂), 3.02-3.12 (m, 4H, 2×CH₂), 3.81(s, 2H, CH₂), 5.56 (d, 1H, J=9.85 Hz, CH), 6.07 (d, 1H, J=16.9 Hz, CH),6.20 (dd, 1H, J=10.1 Hz and 16.9 Hz, CH), 8.07 (bs, 1H, NH), 8.27 (bs,1H, NH). Mass (electrospray+) calculated for C₁₀H₁₇BrN₂O₂ 276 or 278.found 279 (278+H⁺), 299 (276+Na⁺).

Grafting of Primers

The primers were 5′-phosphorothioate oligonucleotides. Grafting wascarried out using 80 μl per channel in 10 mM phosphate buffer pH7 for 1h at RT with the following two oligonucleotides each at a concentrationof 0.5 μM.

P7 non-cleavable primer (supplied by Eurogentec):5′-phosphorothioate-TTTTTTTTTTCAAGCAGAAGACGGCATAC GA-3′OHP5 cleavable primer (supplied by ATD):5′-phosphorothioate-TTTTTTTTTT-(diol)X3-AATGATACG GCGACCACCGA-3′OH

The structure of the diol linker incorporated into the cleavable primerwas as follows:

The channels are washed with 10 mM phosphate buffer, followed by 10 mMTris/10 mM EDTA pH 8.0 (100 μL per channel) to remove unbound DNA.

Colony Formation

An amplification template may be hybridised to the grafted primersimmediately prior to the amplification reaction. The amplificationreaction thus begins with an initial primer extension step rather thantemplate denaturation. Amplification was carried out using a lambdaFtemplate and primers P7 and P5 as shown in FIG. 1.

The hybridization procedure begins with a heating step in a stringentbuffer (95° C. for 5 minutes in TE) to ensure complete denaturationprior to hybridisation of the template. Hybridization was then carriedout in 5×SSC, using template diluted to the desired final concentration.After the hybridization, the chip was washed for 5 minutes with milliQwater to remove salts.

Surface amplification was carried out in an MJ Research thermocycler.

A typical program is as follows:

1—97.5° C. for 0:45 2—X° C. for 1:30 3—73° C. for 1:30

4—Go to 1 [40] times

5—73° C. for 5:00 6—20° C. for 3:00 7—End

Since the first step in the amplification reaction was extension of theprimers bound to template in the initial hybridisation step the firstdenaturation and annealing steps of this program are omitted (i.e. thechip is placed on the heating block only when the amplification mix ispumped through the flow cell and the temperature is at 73° C.)

The annealing temperature (X° C., step 2) depends on the primer pairthat is used. Experiments have determined an optimal annealingtemperature of 57° C. for P5/P7 primers. For other primer-pairs theoptimum annealing temperature can be determined by experiment. Thenumber of cycles may be varied if required.

Amplification was carried out in a reaction solution comprising 1×PCRreaction buffer (supplied with the enzyme) 1M betain, 1.3% DMSO, 200 μMdNTPs and 0.025 U/μL Taq polymerase.

General features of the solid-phase amplification procedure to producenucleic acid colonies are as described in International patentapplications WO 98/44151 and WO 00/18957.

Linearisation

Channels to be cleaved were treated with a solution of 0.1M of sodiumperiodate and 0.1M ethanolamine in water for 1 hour at room temperature.All channels were then washed for 30 minutes with milliQ water at roomtemperature.

Thermal Dehybridisation

Thermal denaturation or de-hybridization of linearised colonies wascarried out in stringent buffer (TE). Temperature was ramped 0.5° C./secto 97.5° C. and held at 97.5° C. for 2 minutes 30 seconds.

Hybridisation of Sequencing Primer

The procedure begins with a heating step in a stringent buffer (TE) toensure complete denaturation of the colonies prior to hybridisation ofthe primer.

Hybridization was carried out in 5×SSC, using an oligonucleotide dilutedto a final concentration of 500 nM.

Typical temperature cycling profile was as follows:

MJ-Research Thermocycler program set:

-   -   (Control method: block)    -   1—0.5° C./sec to 97.5° C.    -   2—97.5° C. for 2:30    -   3—97.5° C. for 0:02        -   —0.1° C. per cycle    -   4—Goto 3 for 574 times    -   5—40° C. for 15:00    -   6—End        5) Long Read Sequencing Protocol on lamdaF Template

Oligo number 562 was hybridised to the linearised clusters prepared asdescribed above at 500 nM. Nucleotide sequences of the template andprimers are shown in FIG. 1.

Sequencing was carried out using modified nucleotides prepared asdescribed in International patent application WO 2004/018493, andlabelled with four different commercially available fluorophores(Molecular Probes Inc.).

A mutant 9° N polymerase enzyme (an exo-variant including the triplemutation L408Y/Y409A/P410V and C223S) was used for the nucleotideincorporation steps.

Enzyme mix (enzymology buffer (50 mM Tris-HCl pH 8.0, 6 mM MgSO4, 1 mMEDTA, 0.05% (v/v) Tween −20) plus 50 μg/ml YAV exo-C223S, and 1 μM eachof the four labelled modified nucleotides) was applied to the clusteredtemplates, typically for 2 min 30 s, and heated to 45° C.

Templates were maintained at 45° C. for 30 min, cooled to 20° C. andwashed for 5 min with enzymology buffer, then 5 min with 5×SSC.Templates were then exposed to one of two imaging buffers:“non-ascorbate buffer” (100 mM Tris pH7.0, 30 mM NaCl), or ascorbatescan buffer (100 mM Tris pH7.0, 30 mM NaCl, 50 mM sodium ascorbate(freshly dissolved, filtered).

Templates were scanned in 4 colours.

Templates were then exposed to cycles of TCEP treatment and enzymologyas follows:

TCEP Treatment

-   -   0.1M Tris pH 7.4 for 390 s    -   Heat to 45° C.    -   TCEP (100 mM in 0.1 M Tris pH 7.4) for 390 s    -   Wait for 30 min, flushing for 20 s every 10 min    -   Cool to 20° C.    -   Enzymology buffer for 390 s    -   5×SSC buffer for 390 s    -   “non ascorbate buffer” (100 mM Tris pH 7.0, 30 mM NaCl) for 330        s OR “ascorbate buffer” (100 mM Tris pH 7.0, 30 mM NaCl, 50 mM        sodium ascorbate) for 330 s    -   9. Scan in 4 colours

Enzymology Cycle

-   -   Enzymology buffer for 390 s    -   Heat to 45° C.    -   Enzyme mix for 390 s    -   Wait for 30 min, flushing for 20 s every 10 min    -   Cool to 20° C.    -   Enzymology buffer for 390 s    -   5×SSC buffer for 390 s    -   “non ascorbate buffer” (100 mM Tris pH 7.0, 30 mM NaCl) for 330        s, scan in 4 colours    -   “DMSO buffer” (100 mM Tris pH 7.0, 30 mM NaCl, 50 mM sodium        ascorbate) for 330 s, scan 4 colours

In total, 25 cycles of data were collected.

A clear difference between the number of clusters detected at each cyclecould be seen. The non ascorbate buffer resulted in a loss of signal foreach cluster, and a loss of the number of detectable clusters, such thatafter seven cycles the sequencing process was no longer working. Usingthe ascorbate buffer, at least 18 cycles of sequencing could beachieved.

The slides were scanned on a white light epifluorescence microscope setup with four separate excitation and emission channels appropriate tothe fluorophores.

Representative results were substantially similar to those illustratedin FIG. 2, demonstrating a clear improvement in brightness in thepresence of ascorbate.

1-4. (canceled)
 5. A method of generating a template for a nucleic acidsequencing reaction comprising, (i) providing a solid support comprisinga plurality of forward amplification primers and a plurality of reverseamplification primers, wherein said plurality of forward amplificationprimers or said plurality of reverse amplification primers comprises acleavage site comprising a diol linkage; (ii) amplifying a templateusing the primers on the support to produce a plurality ofdouble-stranded nucleic acid molecules, wherein both strands of themolecule are attached to the solid support at their 5′ ends; (iii)cleaving one strand of the double stranded molecules at the cleavagesite, (iv) subjecting the cleaved strand to denaturing conditions toremove the portion of the cleaved strand not attached to the solidsupport, thereby generating partially or substantially single-strandedtemplates immobilized to the solid support; (v) hybridizing a sequencingprimer to each single-stranded template and performing a sequencingreaction to determine the sequence of at least one region of thesingle-stranded templates immobilized to the solid support.
 6. Themethod of claim 5, further comprising treating the cleaved strand with acapping agent prior to step (v).
 7. The method of claim 6, whereintreatment with a cleaving agent and treating with a capping agent occurconcurrently.
 8. The method of claim 6, wherein treatment with acleaving agent and treating with a capping agent occur non-concurrently.9. The method of claim 5, wherein cleavage comprises cleaving bytreatment with a chemical agent comprising periodate.
 10. The method ofclaim 5, wherein amplification of the template forms a clustercomprising the plurality of double-stranded molecules.
 11. The method ofclaim 5, wherein the sequencing reaction comprisessequencing-by-synthesis.
 12. The method of claim 5, wherein thesequencing reaction comprises pyrosequencing or sequencing-by-ligation.13. The method of claim 5, wherein the solid support comprises ahydrogel.
 14. The method of claim 13, wherein the hydrogel is apolyacrylamide hydrogel.
 15. The method of claim 13, wherein the solidsupport is prepared by a method comprising polymerizing on the solidsupport a mixture of: (i) a first comonomer which is acrylamide,methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone; and(ii) a second comonomer which is a functionalized acrylamide or acrylateof formula (I):H2C═C(H)—C(═O)-A-B-C  (I); or a methacrylate or methacrylamide offormula (II):H2C═C(CH3)-C(═O)-A-B-C  (II); wherein A is NR or O, wherein R ishydrogen or an optionally substituted saturated hydrocarbyl groupcomprising 1 to 5 carbon atoms; B is an optionally substituted alkylenebiradical of formula —(CHn)- wherein n is an integer from 1 to 50; andwherein n=2 or more, one or more optionally substituted ethylenebiradicals —CH2CH2- of said alkylene biradical may be independentlyreplaced by ethenylene and ethynylene moieties; and wherein n=1 or more,one or more methylene biradicals —CH2- may be replaced independentlywith an optionally substituted mono- or polycyclic hydrocarbon biradicalcomprising from 4 to 50 carbon atoms, or a correspondingdeteromonocyclic or heteropolycyclic biradical wherein at least 1 CH2 orCH2 is substituted by an oxygen sulfur or nitrogen atom or an NH group;and C of A-B-C of formulas (I) or (II) is a group for reaction with acompound to bind said compound covalently to said hydrogel to form apolymerized product, characterized in that polymerization is conductedon and immobilized the polymerized product to a solid support that isnot covalently surface-modified.
 16. A method of generating a templatefor a nucleic acid sequencing reaction comprising, (i) providing a solidsupport comprising a plurality of forward amplification primers and aplurality of reverse amplification primers, wherein said plurality offorward amplification primers or said plurality of reverse amplificationprimers comprises a deoxyuridine (U) at a predetermined cleavage site;(ii) amplifying a template using the primers on the support to produce aplurality of double-stranded nucleic acid molecules, wherein bothstrands of the molecule are attached to the solid support at their 5′ends; (iii) cleaving one strand of the double stranded molecules at thecleavage site, (iv) subjecting the cleaved strand to denaturingconditions to remove the portion of the cleaved strand not attached tothe solid support, thereby generating partially or substantiallysingle-stranded templates immobilized to the solid support; (v)hybridizing a sequencing primer to each single-stranded template andperforming a sequencing reaction to determine the sequence of at leastone region of the single-stranded templates immobilized to the solidsupport.
 17. The method of claim 16, wherein cleavage comprisestreatment with uracil DNA glycosylase (UDG) to generate an abasic siteat the predetermined cleavage site.
 18. The method of claim 17, whereincleavage further comprises treatment with an endonuclease to cleavestrand at the abasic site.
 19. The method of claim 16, wherein the solidsupport comprises a hydrogel.
 20. The method of claim 19, wherein thehydrogel is a polyacrylamide hydrogel.
 21. A method of generating atemplate for a nucleic acid sequencing reaction comprising, (i)providing a solid support comprising a plurality of forwardamplification primers and a plurality of reverse amplification primers,wherein said plurality of forward amplification primers or saidplurality of reverse amplification primers comprises 8-oxo-guanine at apredetermined cleavage site; (ii) amplifying a template using theprimers on the support to produce a plurality of double-stranded nucleicacid molecules, wherein both strands of the molecule are attached to thesolid support at their 5′ ends; (iii) cleaving one strand of the doublestranded molecules at the cleavage site, (iv) subjecting the cleavedstrand to denaturing conditions to remove the portion of the cleavedstrand not attached to the solid support, thereby generating partiallyor substantially single-stranded templates immobilized to the solidsupport; (v) hybridizing a sequencing primer to each single-strandedtemplate and performing a sequencing reaction to determine the sequenceof at least one region of the single-stranded templates immobilized tothe solid support.
 22. The method of claim 21, wherein cleavagecomprises treatment with FPG glycosylsase to generate an abasic site atthe predetermined cleavage site.
 23. The method of claim 22, whereincleavage further comprises treatment with an endonuclease to cleavestrand at the abasic site.
 24. The method of claim 21, wherein the solidsupport comprises a hydrogel.